2 replies to this topic

[edwina]

Hi:
Our lab has bought Invitrogen Flp-In T-rex cells line 1 year ago and we now got our clones. The problem is that the promotor seems to be very strong and our protein aggregated so we want to reduce the expression level. The manuscript of them recommended to use 0.1-1µg/ml Tetracycline or similar conc. of Doxycycline to induced the cell line. Currently we are using 0.1µg/ml. I have asked their technics but they also have no clue and only recommended me to buy their FBS, which is Tetracycline free  
Does anyone has experience with this cell line and do you know till which concentration of Tetracycline can we get half or even less expression level?
Thanks a lot if anyone would like to help!

[scolix]

Try the dialysed FBS, it may help. But no guarantees. It is known that the tet-on system is leaky.

[knuf]

Yeah, we use the same system in our lab and use that same concentration of tetracycline to induce expression, and as far as I understand, there is no way to reduce expression because it uses a high-expression level promoter. If you use a lower concentration, I would guess that it would probably cause expression in a percentage of the cells, as opposed to reduced expression in all of them, but that is just a guess. I have noticed that it takes a while for expression to be at its maximum level. We usually don't see maximum protein level until about a week after induction, so maybe you could work with this aspect of the system? Also, I believe that there are alternative systems with controlled expression (none of which i can recommend or comment on), but that would require starting with a whole new system.